Our HIV vaccine approach is based on initial immunization with a replicating adenovirus (Ad) vector carrying an HIV gene(s) followed by a booster immunization with an HIV envelope protein. The Ad-HIV vaccine replicates in epithelial cells that line mucosal inductive sites, thus eliciting strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We have shown that initial immunizations with an Ad-HIV vaccine also stimulates production of anti-HIV antibodies. Together, the regimen induces strong and durable protective responses. We have demonstrated that administration of Ad-HIV vaccine to the upper respiratory tract as well as to the gut (by oral immunization) elicits memory T cells that possess "homing receptors" leading them to traffic to the intestine, a prime site of HIV infection. Thus the vaccine approach elicits cellular immunity at a location critical for preventing or controlling HIV infection. We are continuing to explore expression of homing receptors following vaccination as surrogate markers of cellular mucosal immunity. Such surrogates will be invaluable when large scale clinical vaccine trials are carried out. It will not be possible to obtain tissue biopsies from large numbers of volunteers, and surrogate markers that can be measured on blood samples will be critical in evaluating the development of mucosal immune responses. Recently we have explored several routes of Ad-recombinant immunization in rhesus macaques including intrarectal, intravaginal, and sublingual as well as the standard administration to the upper respiratory tract intranasally or intratracheally. Interestingly, cellular immune responses as maeasured by interferon-gamma ELISPOT and T cell proliferation assays revealed similar levels of imnmune responses were achieved in peripheral blood by all immunization routes. Administration of an Ad-GFP marker recombinant by the various routes also showed the presence of replicating Ad was similar in rectal tissue and the lung without regard to immunization route. The data suggest use of replicating Ad vectors may facilite vaccine delivery by the easiest, least invasive route. Studies of Tat and Env immunogens in an Ad-recombinant prime/protein boost protocol has shown that the combined immunization regimen elicited enhanced polyfunctional memory cells, both in peripheral blood and in the lung, an easily monitored effector site. Such polyfunctional cells have been associated with better protection. We have also confirmed a protective role for such cells in elite controller macaques. The study of NK cells, key effector cells of innate immunity, is also continuing in order to elucidate their response to Ad-HIV vaccine immunization and their cooperation with vaccine-elicited antibodies in mediating cell killing functions such as antibody-dependent cellular cytotoxicity and antibody-dependent cell mediated viral inhibition. Macaque NK cells have been characterized phenotypically by flow cytometry as a prelude to expanded functional studies. Studies have shown that these activities that span innate and adaptive immunity are correlated with reduced viremia following viral challenge of immunized rhesus macaques. New studies of NKT cells have recently been initiated. We have also investigated host factors that contribute to control of HIV and SIV. Among such factors are particular MHC class 1 alleles. A study in cynomolgus macaques illustrated the importance of typing these alleles in study animals in order to be able to determine whether protective outcomes result from particular vaccine regimens or the possession of protective MHC class I alleles. Overall we have developed expanded capabilities to investigate vaccine-elicited cellular immunity by flow cytometry, as well as by expression of various cytokines and chemokines. Our studies continue to show induction of potent cellular immune responses systemically and mucosally following the replicating Ad-recombinant prime/protein boost regimen.